Two-stage bottom-up tiered approach combining several alternatives for identification of eye irritation potential of chemicals including insoluble or volatile substances

For the assessment of eye irritation, one alternative test may not completely replace the rabbit Draize test. In the present study, we examined the predictive potential of a tiered approach analyzing the results from several alternatives (i.e., the Short Time Exposure (STE) test, the EpiOcular assay, the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) assay and the Bovine Corneal Opacity and Permeability (BCOP) assay) for assessing Globally Harmonized System (GHS) eye irritation categories. Fifty-six chemicals including alcohols, surfactants, and esters were selected with a balanced GHS category and a wide range of chemical classes. From a standpoint of both assessable sample numbers and predictive accuracy, the more favorable tiered approach was considered to be the two-stage bottom-up tiered approach combining the STE test, the EpiOcular assay followed by the BCOP assay (accuracy 69.6%, under prediction rate 8.9%). Moreover, a more favorable predictive capacity (accuracy 71.4%, under prediction rate 3.6%) was obtained when high volatile alcohols/esters with vapor pressures >6 kilopascal (kPa) at 25 °C were evaluated with EpiOcular assay instead of the STE test. From these results, the two-stage bottom-up tiered approach combining the STE test, the EpiOcular assay followed by the BCOP assay might be a promising method for the classification of GHS eye irritation category (Not classified (NC), Category 2 (Cat. 2), and Category 1 (Cat. 1)) for a wide range of test chemicals regardless of solubility.     

Highly accurate predictor of eye irritation utilizing potential parameters of a reconstructed human cornea epithelium model calculated based on Hansen solubility parameters

Concerns regarding animal welfare have led to the need for alternatives to animal eye irritation tests. The reconstructed human cornea-like epithelium (RhCE) test is described in the OECD TG 492 as an alternative to animal eye irritation tests. However, the accuracy and labor investment of this method can be improved if the results can be predicted before the experiment. In this study, we evaluated whether Hansen solubility parameter (HSP) values can be used to predict the results of RhCE method using the LabCyte CORNEA-MODEL for 65 test substances. We found that HSP values can predict the RhCE method with high correlation (accuracy 84.6% (55/65), false-negative rate of 16.2% (7/43), and false-positive rate of 13.6% (3/22). These results indicate that HSP values can be used to predict the results RhCE method using LabCyte CORNEA-MODEL with high reproducibility, and thus are useful for evaluating the safety of substances.     

Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals

Recent changes in regulatory restrictions and social views against animal testing have accelerated development of reliable alternative tests for predicting skin sensitizing potential and potency of many chemicals. Lately, a test battery integrated with different in vitro tests has been suggested as a better approach than just one in vitro test for replacing animal tests. In this study, we created a dataset of 101 test chemicals with LLNA, human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and in silico prediction system. The results of these tests were converted into scores of 0–2 and the sum of individual scores provided the accuracy of 85% and 71% for the potential and potency prediction, compared with LLNA. Likewise, the straightforward tiered system of h-CLAT and DPRA provided the accuracy of 86% and 73%. Additionally, the tiered system showed a higher sensitivity (96%) compared with h-CLAT alone, indicating that sensitizers would be detected with higher reliability in the tiered system. Our data not only demonstrates that h-CLAT can be part of a test battery with other methods but also supports the practical utility of a tiered system when h-CLAT and DPRA are the first screening methods for skin sensitization.     

Exploration and comparison of in vitro eye irritation tests with the ISO standard in vivo rabbit test for the evaluation of the ocular irritancy of contact lenses

In an effort to explore the use of alternative methods to animal testing for the evaluation of the ocular irritancy of medical devices, we evaluated representative contact lenses with the bovine corneal opacity and permeability test (BCOP) and an in vitro eye irritation test using the three-dimensionally-reconstructed human corneal epithelium (RhCE) models, EpiOcular™ and MCTT HCE™. In addition, we compared the obtained results with the ISO standard in vivo rabbit eye irritation test (ISO10993-10). Along with the positive controls (benzalkonium chloride, BAK, 0.02, 0.2, and 1%), the extracts of 4 representative contact lenses (soft, disposable, hard, and colored lenses) and 2 reference lenses (dye-eluting and BAK-coated lenses) were tested. All the lenses, except for the BAK-coated lens, were determined non-irritants in all test methods, while the positive controls yielded relevant results. More importantly, BCOP, EpiOcular™, and MCTT HCE™ yielded a consistent decision for all the tested samples, with the exception of 0.2% BAK in BCOP, for which no prediction could be made. Overall, all the in vitro tests correlated well with the in vivo rabbit eye irritation test, and furthermore, the combination of in vitro tests as a tiered testing strategy was able to produce results similar to those seen in vivo. These observations suggest that such methods can be used as alternative assays to replace the conventional in vivo test method in the evaluation of the ocular irritancy of ophthalmic medical devices, although further study is necessary.     

A tiered approach combining the short time exposure (STE) test and the bovine corneal opacity and permeability (BCOP) assay for predicting eye irritation potential of chemicals

For the evaluation of eye irritation, one in vitro alternative test may not completely replace the Draize test. Therefore, a tiered approach combining several in vitro assays, including cytotoxicity assays, is proposed in order to estimate the eye irritation potential of a wide range of chemical classes. The Short Time Exposure (STE) test, a relatively newer alternative eye irritation test, involves exposing Statens seruminstitut rabbit cornea (SIRC) cells for 5 min to two concentrations (5% and 0.05%) of test material. In the present study, we examined the predictive capacity of a tiered approach analyzing the results from the STE test and then the results of the bovine corneal opacity and permeability (BCOP) assay for assessing globally harmonized system (GHS) eye irritation rankings of various chemicals. The accuracy of predicting the GHS rankings was slightly improved when the tiered approach combination of STE test and BCOP assay was used compared to when the STE test irritation rank classification was used alone. Moreover, the under prediction rate was substantially improved when this tiered approach was used. From these results, the tiered approach of combining the data analysis of the STE test and BCOP assay might be a promising alternative eye irritation test strategy.     

Expansion of the application domain of a macromolecular ocular irritation test (OptiSafe™)

The OptiSafe (OS) test is shelf-stable, macromolecular eye irritation test that does not include any animal ingredient or component (“vegan”). The purpose of this study was to evaluate the test's accuracy for an expanded application domain for both the original and recently updated OS method. This study involved the testing of additional ocular corrosives and previously excluded foaming agents (“surfactants”) using both the original and updated OS methods and then combining these data with prior validation data for a total of 147 chemicals. Predictivity was evaluated by a statistical comparison of the OptiSafe predictions with historical in vivo “Draize” rabbit eye data for the same chemicals (from public databases). We report that for the detection of chemicals not requiring classification for eye irritation [Globally Harmonized System of Classification and Labeling of Chemicals (GHS) No Category], the accuracy, specificity, and sensitivity were 92.8%, 79.6%, and 100.0%, respectively, for the updated method; for the detection of chemicals inducing extreme eye damage/corrosion (GHS Category 1), the accuracy, specificity, and sensitivity were 79.4%, 71.8%, and 91.7%, respectively, for the updated method. Results indicate that both the original and updated methods have a high accuracy for the expanded application domain that included ocular corrosives and surfactants.     

Development of the SkinEthic HCE Time-to-Toxicity test method for identifying liquid chemicals not requiring classification and labelling and liquids inducing serious eye damage and eye irritation

This study describes the development of a Time-to-Toxicity approach for liquids (TTL) based on the SkinEthic™ HCE tissue construct, capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (No Cat.) from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (Cat. 1).Briefly, the Time-to-Toxicity of 56 liquids was evaluated by exposing SkinEthic™ HCE tissue constructs to the test chemical for three different time periods (5-min, 16-min, and 120-min). Based on the viability observed for the different exposure periods, a classification was assigned. The within laboratory reproducibility in terms of concordance in classifications (3 UN GHS categories), based on a set of 50 liquids, was 80.0%. Furthermore, 84.3% Cat. 1 (N = 17), 79.4% Cat. 2 (N = 21) and 72.2% No Cat. (N = 18) were correctly identified with the SkinEthic™ HCE TTL test method. This study provides evidence that the SkinEthic™ HCE Time-to-Toxicity method (multiple exposure times) is capable of distinguishing Cat. 2 liquids from Cat. 1 liquids. This is an advantage compared to the SkinEthic™ HCE EITL method (single exposure time) that can distinguish No Cat. chemicals from chemicals that do require classification and labelling for eye irritation/serious eye damage (Cat. 2/Cat. 1).     

Second-phase validation study of short time exposure test for assessment of eye irritation potency of chemicals

A Short Time Exposure (STE) test is a cytotoxicity test that uses SIRC cells (rabbit corneal cell line) to assess eye irritation potency following a 5-min chemical exposure. This second-phase validation study assessed the predictive capacity of the STE test using 40 coded test substances at three laboratories. A Validation Management Team (VMT) then evaluated the predictivity of the STE test for United Nation (UN) Globally Harmonized System (GHS) categories using 63 test substances including the results of the first-phase validation study.The STE test can assess not only the severe or corrosive ocular irritants (corresponding to the UN GHS Category 1) but also non-irritant (corresponding to UN GHS Non Category) from other toxicity classes, especially for limited types of test substances. The predictivity by STE test, however, was insufficient for identification of UN GHS categories (Category 1, Category 2, or Non Category).These results suggest that the STE test can be recommended as an initial step in a top-down approach to identification of severe irritants and test substances that require classification for eye irritation (UN GHS Category 1) as well as an initial step in a bottom-up approach to identification of test substances that do not require classification for eye irritation (UN GHS Non Category) from other toxicity classes, especially for limited types of test substances. On the other hand, the STE test is not considered adequate for the identification of mild or moderate irritants (i.e., UN GHS Categories 2A and 2B) and severe irritants (UN GHS Category 1).     

The EyeIRR-IS assay: Development and evaluation of an in vitro assay to measure the eye irritation sub-categorization of liquid chemicals

Several alternative methods have been developed and regulatory adopted by OECD as in vitro alternatives to the Draize eye irritation assay either to detect chemicals not requiring classification (No Category) or inducing serious damage to the eye (Category 1) but none are sensitive enough to identify chemicals inducing reversible eye effects (category 2) which are categorised by default. Therefore, the discriminatory power of a genomic approach applied to the SkinEthic™ Human Corneal Epithelium (HCE) model was investigated to allow subcategorization capacity according to UN GHS classification. An algorithm based on gene expression modulation on a training (62) and a test (31 liquids) chemical set, tested neat and at 30%was evaluated in an assay called EyeIRR-IS. Its accuracy prediction to distinguish Cat1/Cat2 from No Cat was 95% with a specificity of 89% and a sensitivity of 98%. For subcategorization into the 3 GHS classes the accuracy reached 84% with 94% Cat1, 67% Cat2 and 89% No Cat correctly predicted. No Cat.1 chemicals were underestimated as negative with a majority of misclassified Cat2 over predicted as Cat 1. In conclusion, the performance of the assay suggests its added value in a defined approach for liquids to replace the Draize assay.     

SkinEthic HCE Time-to-Toxicity on solids: A test method for distinguishing chemicals inducing serious eye damage,eye irritation and not requiring classification and labelling

This study describes the development of a Time-to-Toxicity approach for solids (TTS) based on the SkinEthic™ HCE tissue construct, capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (No Cat.) from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (Cat. 1).Briefly, the time-to-toxicity of 69 solids was evaluated by exposing SkinEthic™ HCE tissue constructs to the test chemical for two different time periods (30-min, and 120-min). Based on the viability observed for the different exposure periods, a classification was assigned. The within laboratory reproducibility in terms of concordance in classifications (3 UN GHS categories), based on a set of 48 solids, was 93.7%. Furthermore, 73.6% Cat. 1 (N = 24), 55.6% Cat. 2 (N = 15) and 72.2% No Cat. (N = 30) were correctly identified with the SkinEthic™ HCE TTS test method. This study provides evidence that the SkinEthic™ HCE Time-to-Toxicity method (multiple exposure times) can distinguish Cat. 2 solids from Cat. 1 solids. This is an added value compared to the SkinEthic™ HCE EITS method (single exposure time) that can distinguish No Cat. chemicals from chemicals that do require classification and labelling for eye irritation/serious eye damage (Cat. 2/Cat. 1).     

Sub-categorisation of skin corrosive chemicals by the EpiSkin™ reconstructed human epidermis skin corrosion test method according to UN GHS: Revision of OECD Test Guideline 431

The EpiSkin™ skin corrosion test method was formally validated and adopted within the context of OECD TG 431 for identifying corrosive and non-corrosive chemicals. The EU Classification, Labelling and Packaging Regulation (EU CLP) system requires the sub-categorisation of corrosive chemicals into the three UN GHS optional subcategories 1A, 1B and 1C. The present study was undertaken to investigate the usefulness of the validated EpiSkin™ test method to identify skin corrosive UN GHS Categories 1A, 1B and 1C using the original and validated prediction model and adapted controls for direct MTT reduction. In total, 85 chemicals selected by the OECD expert group on skin corrosion were tested in three independent runs. The results obtained were highly reproducible both within (>80%) and between (>78%) laboratories when compared with historical data. Moreover the results obtained showed that the EpiSkin™ test method is highly sensitive (99%) and specific (80%) in discriminating corrosive from non-corrosive chemicals and allows reliable and relevant identification of the different skin corrosive UN GHS subcategories, with high accuracies being obtained for both UN GHS Categories 1A (83%) and 1B/1C (76%) chemicals. The overall accuracy of the test method to subcategorise corrosive chemicals into three or two UN GHS subcategories ranged from 75% to 79%. Considering those results, the revised OECD Test Guideline 431 permit the use of EpiSkin™ for subcategorising corrosive chemicals into at least two classes (Category 1A and Category 1B/1C).     

Validation study of a new reconstructed human epidermis model EPiTRI for in vitro skin irritation test according to OECD guidelines

Following the global trend of reducing animal testing, various reconstructed human epidermis (RHE) models for skin irritation test (SIT) have been developed, verified, validated and included in OECD TG 439. We developed a new RHE called EPiTRI and a SIT method using EPiTRI (EPiTRI-SIT model) following the OECD guidelines. EPiTRI possesses morphological, biochemical and physiological properties similar to human epidermis with well-differentiated multilayered viable cells with barrier function. The EPiTRI-SIT model was tested for 20 reference chemicals in Performance Standard of OECD TG 439 (GD 220), showing good predictive capacity with 100% sensitivity, 70% specificity and 85% accuracy. EPiTRI had sensitivity in detecting di-n-propyl disulphate, as an irritant chemical (UN GHS Category 2), whereas most validated reference methods detected it as a non-irritant. An international validation study of EPiTRI-SIT was conducted in four laboratories to confirm the within- and between-laboratory reproducibility, as well as predictive capacity. The phase I/II within-laboratory and between-laboratory reproducibility was 100%/95% and 95%, respectively. The overall sensitivity, specificity and accuracy of EPiTRI-SIT was 96%, 70% and 83%, respectively, which fulfilled the OECD criteria. Thus, EPiTRI, meets the criteria of Performance Standards of OECD TG 439 (GD 220) and is suitable for screening irritating chemicals in vitro.     

Assessment of skin and eye irritation of 14 products under the stepwise approach of the OECD

The determination of acute eye and skin irritation is included in international regulatory requirements for the testing of chemicals, because of the possibility of exposure during the production, transport, marketing, and disposal of products. Although there have been some advances in the areas of refinement and reduction, no single battery of tests has emerged as being acceptable as a complete replacement for the conventional Draize rabbit eye and skin irritation tests. Currently, dermal irritation and ocular irritation are generally evaluated in a sequential manner in the context of tiered assessment strategies. In this work, we show how 14 products, mostly designed to be used in agriculture, were evaluated in the Center of Experimental Toxicology of the Center for the Production of Laboratory Animals (Centro Nacional para la Producción de Animales de Laboratorio; CENPALAB) in order to assess their acute dermal and ocular effects. The performed studies include the acute dermal toxicity test, the acute dermal irritation/corrosion test, the hen's egg test-chorioallantoic membrane (HET-CAM) method, and the acute eye irritation/corrosion test. In general, it could be concluded that of the 14 products assessed, none of them showed systemic effects, but local reactions mainly to the eyes. The most significant effects were apparently related to the effects of azadirachtin, an active principle of 2 tested neem derivatives.     

Development of an in vitro corrosion/irritation prediction assay using the EpiDerm skin model.

A validation of the in vitro skin corrosion method using the EpiDerm skin model was performed using 12 recommended chemicals. All chemicals were correctly classified by OECD test guideline 431. In order to predict corrosion and/or irritation potential, additional compound exposure times and IL-1alpha measurements were included in a tiered testing approach. Four exposure times were performed followed by MTT (viability) and IL-1alpha measurement. This allowed classification of corrosive chemicals (OECD guideline 431) and those likely to be severe irritants. If the chemical was found to be corrosive or a severe irritant, no further experimental work was performed, otherwise a second experiment was performed using three further exposure times (same endpoints). The second experiment provided information on whether the chemical was likely to be a moderate/mild irritant. If the chemical was negative following both experiments, it was predicted as non-corrosive/non-irritating. A total of 12 chemicals were tested in the irritation or combined assay (five non-irritants, seven irritants). Specificity (% non-irritants concurring with EU classification) was 60% (MTT) and 100% (MTT+IL-1alpha). Sensitivity (% irritants concurring with EU classification) was 86% (MTT) and 86% (MTT+IL-1alpha). Accuracy (% chemicals correctly identified) was 75% (MTT) and 92% (MTT+IL-1alpha).     

Suitability of histopathology as an additional endpoint to the Isolated Chicken Eye Test for classification of non-extreme pH detergent and cleaning products

A.I.S.E. investigated the suitability of histopathological evaluations as an additional endpoint to the regulatory adopted ICE in vitro test method (OECD TG 438) to identify non-extreme pH detergent and cleaning products that require classification as EU CLP/UN GHS Category 1 (serious eye damage). To this aim, a total of 30 non-extreme pH products covering the range of in vivo classifications for eye irritation, and representing various product categories were tested. Epithelium vacuolation (mid and lower layers) and erosion (at least moderate) were found to be the most relevant histopathological effects induced by products classified in vivo as Category 1. Histopathology criteria specifically developed for non-extreme pH detergent and cleaning products were shown to correctly identify materials classified as Category 1 based on in vivo persistent effects, and to significantly increase the overall sensitivity of the standard ICE prediction model for Category 1 identification (to 75%) whilst maintaining a good concordance (73%). In contrast, use of EU CLP additivity approach for classification of mixtures was considerably less predictive, with a concordance of only 27%, and 100% over-predictions of non-Category 1 products. As such, use of histopathology as an addition to the ICE test method was found suitable to identify EU CLP/UN GHS Category 1 non-extreme pH detergent and cleaning products and to allow a better discrimination from Category 2 products.     

A new 3D reconstituted human corneal epithelium model as an alternative method for the eye irritation test

Many efforts are being made to develop new alternative in vitro test methods for the eye irritation test. Here we report a new reconstructed human corneal epithelial model (MCTT HCE model) prepared from primary-cultured human limbal epithelial cells as a new alternative in vitro eye irritation test method. In histological and immunohistochemical observation, MCTT HCE model displayed a morphology and biomarker expressions similar to intact human cornea. Moreover, the barrier function was well preserved as measured by high transepithelial electrical resistance, effective time-50 for Triton X-100, and corneal thickness. To employ the model as a new alternative method for eye irritation test, protocol refinement was performed and optimum assay condition was determined including treatment time, treatment volume, post-incubation time and rinsing method. Using the refined protocol, 25 reference chemicals with known eye irritation potentials were tested. With the viability cut-off value at 50%, chemicals were classified to irritant or non-irritant. When compared with GHS classification, the MCTT HCE model showed the accuracy of 88%, sensitivity of 100% and specificity of 77%. These results suggest that the MCTT HCE model might be useful as a new alternative eye irritation test method.     

An evaluation of a cultured human corneal epithelial tissue model for the determination of the ocular irritation potential of pharmaceutical process materials

Irritation and other forms of local toxicity following contact with eyes is a potentially serious problem arising from occupational exposure to chemicals. Traditionally, evaluation of the irritant potential of novel chemicals has relied on the use of in vivo studies with rabbits. Concerns about the predictive potential of in vivo methods for human hazard and demand for economical and rapid screening of chemicals has stimulated a great deal of work to investigate in vitro alternatives for evaluating ocular irritation potential. This publication describes a screening study to assess a reconstituted corneal epithelial culture system, as an alternative for testing for ocular irritation with pharmaceutical process materials, extending the chemical domain with which this system has been tested. A total of 21 test chemicals were applied to commercially supplied reconstituted human corneal epithelial (HCE) cultures and effects on tissue viability (MTT reduction assay), tissue histology and IL-alpha expression were assessed. Positive controls (0.5% and 1% SDS) showed dose- and time-related adverse effects on tissues, consistent with known irritant effects. Negative controls showed no histological changes and retained high viability throughout the time-course of the experiment. Concordance was excellent with accuracy at each sampling time point of over 80% when viability (MTT reduction) was compared with existing EU classification of the test articles for ocular irritation (classification based on results of in vivo evaluation). Tissue viability as estimated by MTT reduction appears most useful as the primary means of assessing the irritation potential of the chemicals. Histopathological examination generally agreed with the results of the MTT assay. However, the use of cytokine analysis will need further consideration as results for this parameter showed no relationship with known irritation potential. These results infer that HCE cultures, alone or as a part of a tiered hazard screening programme, have promise for use in reducing reliance on live subject tests and contribute to generation of an appropriate hazard classification and label advice.     

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: Skin irritation

Evaluation of the skin irritancy and corrosivity potential of an ingredient is a necessity in the safety assessment of cosmetic ingredients. To date, there are two formally validated alternatives to the rabbit Draize test for skin corrosivity in place, namely the rat skin transcutaneous electrical resistance (TER) assay and the Human Skin Model Test using EpiSkin™, EpiDerm™ and SkinEthic™ reconstructed human epidermal equivalents. For skin irritation, EpiSkin™, EpiDerm™ and SkinEthic™ are validated as stand-alone test replacements for the rabbit Draize test. Data from these tests are rarely considered in isolation and are evaluated in combination with other factors to establish the overall irritating or corrosive potential of an ingredient. In light of the deadlines established in the Cosmetics Directive for cessation of animal testing for cosmetic ingredients, a COLIPA scientific meeting was held in Brussels on 30th January, 2008 to review the use of alternative approaches and to set up a decision tree approach for their integration into tiered testing strategies for hazard and safety assessment of cosmetic ingredients and their use in products. In conclusion, the safety assessments for skin irritation/corrosion of new chemicals for use in cosmetics can be confidently accomplished using exclusively alternative methods.     

Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model,LabCyte CORNEA-MODEL

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1 min for liquid chemicals or 24 h for solid chemicals, and the post-exposure incubation periods were set at 24 h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test.     

Two novel prediction models improve predictions of skin corrosive sub-categories by test methods of OECD Test Guideline No. 431

Alternative test methods often use prediction models (PMs) for converting endpoint measurements into predictions. Two PMs are used for the skin corrosion tests (SCTs) of the OECD Test Guideline No. 431 (TG 431). One is specific to EpiSkin™ test method, whereas EpiDerm™, SkinEthic™ RHE and epiCS® share a common PM. These methods are based on reconstructed human epidermis models wherein cell viability values are measured. Their PMs allow translating those values into sub-categories of corrosive chemicals, Category 1A (Cat1A) and a combination of Categories 1B/1C (Cat1BC), and identifying non-corrosive (NC) chemicals. EpiSkin™'s PM already results in sufficiently accurate predictions. The common PM of the three others accurately identifies all corrosive chemicals but, for sub-categorization, an important fraction of Cat1BC chemicals (40–50%) is over-predicted as Cat1A. This paper presents a post-hoc analysis of validation data on a set of n = 80 chemicals. It investigates: why this common PM causes these over-predictions and how two novel PMs that we developed (PMvar1 and PMvar2) improve the predictive capacity of these methods. PMvar1 is based on a two-step approach; PMvar2 is based on a single composite indicator of cell viability. Both showed a greater capacity to predict Cat1BC, while Cat1A correct predictions remaining at least at the same level of EpiSkin™. We suggest revising TG 431, to include the novel PMs in view of improving the predictive capacity of its SCTs.